Peroxisome proliferator-activated receptor ɣ agonist mediated inhibition of heparanase expression reduces proteinuria

Summary Background Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor ɣ (PPARɣ) agonists can ameliorate proteinuria. Since a recent study showed that PPARɣ regulates HPSE expression in liver cancer cells, we hypothesized that PPARɣ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. Methods Regulation of HPSE by PPARɣ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARɣ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARɣ agonist pioglitazone. Findings Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARɣ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARɣ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. Interpretation PPARɣ-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. Funding This study was financially supported by the 10.13039/501100002997Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by 10.13039/100016036Top Sector Life Sciences & Health to the 10.13039/501100002997Dutch Kidney Foundation to stimulate public-private partnerships).


Introduction
Proteinuria is one of the first clinical signs of many glomerular diseases and an independent risk factor for the progression to renal failure. 1 Proteinuria can be caused by damage to any of the 3 layers of the glomerular filtration barrier (GFB), which is composed of a fenestrated endothelium covered with a glycocalyx, the glomerular basement membrane and podocytes. All layers of the GFB should be intact to prevent the development of proteinuria. 2 Peroxisome proliferator-activated receptor ɣ (PPARɣ) is a transcription factor that belongs to the superfamily of nuclear receptors. Upon stimulation, PPARɣ forms a heterodimer with the retinoid X receptor, and this heterodimer regulates the transcription of various target genes. 3 PPARɣ has a large binding pocket that enables it to interact with naturally occurring and synthetic ligands of great structural variety. A group of synthetic, high affinity agonists for PPARɣ are the thiazolidinediones (TZDs), which include pioglitazone and rosiglitazone. Initially, TZDs were developed to reduce insulin resistance and thereby to treat type 2 diabetes. 4,5 However, a number of studies also suggested that PPARɣ agonists have direct renoprotective effects in experimental diabetes. [6][7][8] Moreover, PPARɣ agonists have also been suggested to be renoprotective in several (experimental) non-diabetic glomerular diseases, for example, human focal segmental glomerulosclerosis. [9][10][11][12][13] A recent study showed that PPARɣ agonists reduced heparanase (HPSE) gene transcription in hepatocellular carcinoma metastases by direct binding of PPARɣ to the HPSE promoter. 14 HPSE is the only mammalian enzyme that can cleave negatively charged heparan sulfate (HS), 15 and loss of HS in the GFB has been associated with the development of proteinuria. 16, 17 We previously showed that HPSE is essential for the development of proteinuria and subsequent renal damage in experimental glomerulonephritis and diabetic nephropathy. 18,19 Furthermore, several studies showed reduced proteinuria and improved renal function after inhibition of HPSE activity in glomerular diseases including experimental diabetic nephropathy, glomerulonephritis, anti-GBM antibody disease, and passive Heymann nephritis. 18,[20][21][22][23] We hypothesized that PPARɣ agonists exert their renoprotective effect by inhibiting the expression of glomerular HPSE since HPSE is essential for the development of proteinuria, TZDs reduce proteinuria, and PPARɣ regulates HPSE expression in liver cancer cells. In the current study, we evaluated the effects of PPARɣ agonism and antagonism on glomerular HPSE and HS expression in vivo and in vitro. We evaluated whether PPARɣ directly regulates HPSE transcription. Finally, we assessed the plasma HPSE activity in type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARɣ agonist pioglitazone.

Research in context
Evidence before this study The heparan sulfate-degrading enzyme, heparanase, is essential for the degradation of glomerular endothelial glycocalyx and the development of proteinuria in glomerular diseases. Thiazolidinediones are synthetic, high affinity agonists for the nuclear transcription factor, peroxisome proliferator-activated receptor ɣ (PPARɣ), which ameliorates proteinuria in certain glomerular diseases.
Added value of this study This paper describes that heparanase is regulated by PPARy both in vitro, in cultured glomerular cells, and in vivo, in a rat model for focal segmental glomerulosclerosis. In humans treated with pioglitazone, plasma heparanase activity tended to be decreased. It is demonstrated that PPARɣ directly binds to the heparanase promotor region in both podocytes and glomerular endothelial cells.
Aldrich) or vehicle via an intraperitoneal injection. After 6 weeks, rats were sacrificed. In addition, healthy Wistar rats were treated with daily intraperitoneal injections with 2.5 mg/kg body weight of the PPARɣ antagonist GW9662 (Sigma-Aldrich) or vehicle, as previously described. 11 Rats were sacrificed after 3 weeks.

Immunofluorescence staining
Glomerular expression of HPSE and HS was determined by immunofluorescence staining as described previously. 29 Primary antibodies included the polyclonal anti-HPSE antibody HPA1 (ProsPecTany, Rehovot, Israel (RRID:AB_2246577)) and the VSV-tagged single chain HS variable fragment (scFv) antibody EV3B2 (N-, and 6-O sulfation). 30 Secondary antibodies included goat anti-rabbit IgG Alexa 488 (Invitrogen Life Technologies, RRID:AB_143165) for detection of HPSE and anti-VSV Cy3 (Sigma-Aldrich, RRID:AB_259043) for detection of EV3B2. Staining intensities of HPSE and HS were scored in fifty glomeruli per section on a scale between 0 (no staining) and 10 (maximal staining intensity). Scoring was performed on blinded sections by two independent investigators.

RNA isolation and real-time PCR
Total RNA was isolated from rat renal cortex, mPC-5 and mGEnC-1 using the RNeasy mini kit (Qiagen), according to manufacturer's instructions. 1 μg of RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany). HPSE mRNA expression was determined by real-time PCR on the CFX real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green Supermix (Roche Diagnostics) and gene-specific primers (Table 1; Isogen Life Science, de Meern, the Netherlands). Relative HPSE mRNA expression was determined using the delta-delta C T method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene.

Heparanase activity assay
Renal cortical HPSE activity was determined by a commercially available assay (AMS Biotechnology, Abingdon, UK, cat no# Ra001-02-K) following manufacturer's instructions. Plasma HPSE activity was determined by a commercially available heparan degrading enzyme assay kit (Takara Bio, Shiga, Japan, cat no# MK412) according to the manufacturer's instructions.
Heparanase protein assay HPSE protein in plasma was measured using a human heparanase ELISA kit (Abcam, Cambridge, UK, cat. no. #ab256401) according to the manufacturers instruction.
Transendothelial albumin passage mGEnC-1 were seeded on polyester membranes in tissue culture inserts (Corning Incorporated, NY, USA). After differentiation, cells were treated with adriamycin in the presence or absence of the PPARɣ agonist pioglitazone, or with the PPARɣ antagonist GW9662 as outlined above. Transendothelial albumin passage was determined as described previously. 31 Luciferase reporter assay OK cells transfected with a pGL3 firefly luciferase vector containing the 3.5 kb promoter region of the human HPSE gene 32 or an empty pGL3 vector construct, were treated with 10 μM GW9662 (Sigma-Aldrich). The pRL-CMV construct (Promega Corp., Fitchburg, WI) was used to correct for transfection efficiency. OK cells were harvested 24 h after transfection and luciferase activity was determined using the Dual-Luciferase reporter assay (Promega) according to the manufacturer's instructions.
Chromatin immunoprecipitation assay to determine PPARɣ binding to the HPSE promoter Chromatin was cross-linked using 1.5 mM ethylene glycol bis (succinimidyl succinate) (EGS, Thermofischer Scientific) and 1% formaldehyde (Sigma) in phosphate buffered saline (PBS). Cells were scraped, the cell suspensions were collected and centrifuged at 2500 rpm for 4 min. The pellet, containing the cells was stored at −80 • C until further use. After thawing, nuclei were isolated with subsequent lysis buffer 1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10%

Target gene
Primer sequence  glycerol, 0.5% NP-40, 0.25% Triton-X-100) supplemented with freshly added proteinase inhibitor cocktail (1:100, v/v), followed by centrifugation at 2000 × g for 5 min. Nuclei were dissolved in lysis buffer 2 (10 mM Tris HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) supplemented with freshly added phosphatase inhibitor, followed by centrifugation at 2000 × g for 5 min. Afterwards, the pellet was dissolved in lysis buffer 3 (10 mM Tris HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1% NP-40) supplemented with freshly added phosphatase inhibitor. The chromatin was sonicated using a Bioruptor (Diagenode, Seraing, Belgium) with 10 cycles of 30 ′ ON/OFF. 1% (v/v) Triton-X-100 was added to the sonicated samples and the samples were centrifuged on full speed at 4 • C for 10 min and the pellet discarded. Immunoprecipitation was performed with 1.07 μg of PPARɣ antibody (Genetex) or IgG control (Abcam ab46540) coupled to 30 μl of Dynabeads Protein-A (Invitrogen). 5 μg of pre-cleared chromatin was added to the antibody-beads complexes and incubated at 4 • C with rotation overnight. Beads were washed with RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA, 0.7% Nadeoxycholate, 1% NP-40, 0.5M LiCl) and afterwards with TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, pH8.0). The antigen-antibody complexes were eluted with elution buffer (1% SDS, 0.1M NaHCO3) at 62 • C for 4 h. Input samples served as controls. Eluted samples were incubated with 2 μl RNAse (10 mg/ml stock, Thermo Scientific) at 37 • C for 1 h. Next, 4 μl of Proteinase K (20 mg/ml stock, Roche Diagnostics, Germany) was added to the samples and incubated at 55 • C for 2 h. DNA fragments were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturers' instructions. DNA enrichment was analyzed by real-time PCR using a forward and reverse primer for the predicted PPARɣ-binding site in the promoter of HPSE ( Table 2). The enrichment of the promoter sequences in the DNA samples was calculated relative to the percentage of input.

Patients and study design
Patient plasma samples (n = 38) were obtained from two separate studies. 34 patient plasma samples were obtained from the PIRAMID (Pioglitazone Influence on tRiglyceride Accumulation in the Myocardium In Diabetes) study, which was a 24-week prospective, randomized, double-blind, double-dummy with active comparator, 2-center parallel-group intervention. 33 Males 45 to 65-year old with uncomplicated T2DM were eligible. The inclusion criteria were a glycohemoglobin level of 6.5%-8.5% at screening, body mass index [weight/(length 2 )] of 25-32 kg/m 2 , and blood pressure below 150/85 mm Hg. The exclusion criteria were any clinically significant disorder, particularly any history or complaints of cardiovascular or liver disease or diabetes-related complications, and prior use of thiazolidinediones or insulin. Study procedures are described previously. 33 In short, patients received pioglitazone (15 mg once daily, titrated to 30 mg once daily after 2 weeks) and underwent outcome measurements at baseline and at study termination after 24 weeks. Four patient plasma samples were obtained from a 16-week phase IIIB multicenter randomized double-blind study. 34,35 Inclusion and exclusion criteria of this study were similar to the criteria described for the PIRAMID study. Study procedures are described previously. 34,35 In short, patients received pioglitazone (30 mg once daily), and outcome measurements were obtained at baseline and at study termination after 16 weeks.

Statistical analysis
Data is presented as mean ± SEM. Significance was evaluated by a one-way ANOVA and post hoc analysis with the Tukey's multiple comparison test. A student's t-test was used to evaluate differences between two groups. Significance in transendothelial albumin passage was evaluated by a 2-way repeated measures ANOVA with a Bonferroni post-test. In the PPARɣ-ChIP, outliers were identified using a ROUT-test with a Q-value of 5%.

Role of funders
The funding sources had no role in study design, data collection, data analysis, interpretation and writing of this manuscript.

Results
Adriamycin-induced heparanase expression is attenuated by the PPARɣ agonist pioglitazone To study the effect of PPARɣ agonism on glomerular HPSE and HS expression in vivo, adriamycin nephropathy (AN; an animal model for human FSGS) was induced in rats that were subsequently treated with the PPARɣ agonist pioglitazone or vehicle for 6 weeks. Induction of AN resulted in the development of proteinuria, which was significantly reduced by treatment with pioglitazone, as we described previously. 11 Cortical HPSE mRNA expression (Fig. 1a), glomerular HPSE protein expression (Fig. 1b and e), and cortical HPSE activity (Fig. 1c) were significantly increased by induction of AN. Daily treatment with pioglitazone normalized HPSE expression and activity (Fig. 1a-c and e). Moreover, glomerular HS expression was significantly reduced by induction of AN but preserved by treatment with pioglitazone ( Fig. 1d and f).

The PPARɣ agonist pioglitazone reduced HPSE expression and transendothelial albumin passage in vitro
To extend the in vivo effects of the PPARɣ agonist pioglitazone on glomerular HPSE expression in AN, we evaluated whether pioglitazone regulates HPSE expression in cultured mGEnC-1, mPC-5, ciGEnC and hPOD. Cell stimulation with adriamycin significantly induced HPSE mRNA expression in mouse podocytes, whereas HPSE mRNA expression was reduced by treatment with pioglitazone (Fig. 2a). Stimulation of mouse glomerular endothelial cells with adriamycin reduced HPSE mRNA expression, which was not affected by treatment with pioglitazone (Fig. 2b). Notably, HS expression on mGEnC-1 was reduced by stimulation with adriamycin. 31 At the functional level, transendothelial albumin passage was significantly increased by stimulation with adriamycin, which was reduced by additional treatment with pioglitazone (Fig. 2c). Human glomerular endothelial cells did upregulate HPSE mRNA expression upon adriamycin stimulation, which was ameliorated by treatment with pioglitazone (Fig. 2d). On the contrary, human podocytes show no response to adriamycin on HPSE mRNA expression, which might indicate that hPODs are not a good model system for this study (data not shown).

Pharmacologic inhibition of PPARɣ induces heparanase expression and activity in vivo
Healthy rats were treated daily with the irreversible PPARɣ antagonist GW9662 for 3 weeks to study the effects of pharmacologic inhibition of PPARɣ on glomerular HPSE and HS expression in vivo. As we described previously, treatment with GW9662 induced significant proteinuria. 11 Cortical HPSE mRNA expression (Fig. 3a), glomerular HPSE protein expression ( Fig. 3b and e), and cortical HPSE activity (Fig. 3c) were increased by treatment with GW9662, although this was not significant for HPSE protein expression (P = 0.09).
In line, glomerular HS expression was reduced by treatment with GW9662 ( Fig. 3d and f).
The PPARɣ antagonist GW9662 induces transendothelial albumin passage in vitro in a heparanase-dependent manner Cultured mGEnC-1, mPC-5, ciGEnC and hPOD were treated with 1 or 10 μM of the PPARɣ antagonist GW9662 for 24 h to evaluate the effects of PPARɣ antagonism on glomerular HPSE expression in vitro.
On the contrary, human podocytes and endothelial cells did not up-regulate HPSE mRNA expression upon stimulation with GW9662 (data not shown).

The PPARɣ antagonist GW9662 increased heparanase promoter activity
A luciferase reporter assay was performed to evaluate whether the in vivo and in vitro observed effects of the PPARɣ antagonist GW9662 on HPSE expression are caused by a direct regulation of HPSE promoter activity. 32 Treatment with 10 μM GW9662 significantly induced HPSE promoter activity (Fig. 5a).

Direct binding of PPARɣ to the HPSE promoter is regulated by adriamycin and PPARɣ agonists and antagonists
Next, a Chromatin Immunoprecipitation (ChIP) assay was performed to evaluate whether the observed effects of adriamycin, PPARɣ agonist pioglitazone, and PPARɣ antagonist GW9662 on HPSE expression in mPC-5 and mGEnC-1 cells were mediated by direct binding of PPARɣ to the endogenous HPSE promoter. Within the region of 2000 bases before and after the HPSE transcription start site (TSS), three putative PPARɣ binding sites (PPRE's) can be identified using MotifMap, 36 i.e at a distance of −1667, −436, and +1007 bases. Binding of PPARɣ to the HPSE promoter was evaluated in cultured mPC-5 and mGEnC-1 treated with adriamycin either with or without pioglitazone, or with the PPARɣ antagonist GW9662, and in untreated cells. In mPC-5 exposed to adriamycin, binding of PPARɣ to the HPSE promoter was significantly reduced for binding site −436 and +1007, whereas there was a similar trend for the binding site −1667. Treatment with pioglitazone prevented the adriamycin-induced decline in PPARɣ binding to all 3 PPARɣ binding sites in the HPSE promoter. However, treatment with GW9662 did not reveal a significant reduction in PPARɣ binding to the PPARɣ binding sites in the HPSE promoter, although there was a trend for lower PPARɣ binding for binding sites −436 and −1667 (Fig. 5b). In mGEnC-1, the different treatments did not significantly change PPARɣ binding to the HPSE promoter for all 3 binding sites. Nevertheless, there is a trend for adriamycin-induced lowering of PPARɣ binding for binding sites −1667 and +1007, as well as for GW9662-induced lowering of PPARɣ binding for all three binding sites (Fig. 5c).

Pioglitazonde tends to reduce the plasma HPSE activity in type 2 diabetes mellitus patients
Full clinical characterization is described in the original manuscripts of the studies from which the samples have been obtained. [33][34][35] The most important clinical characteristics of the patients are summarized in Table 3. Overall, hemoglobin A1c (HbA 1C ), plasma glucose, and diastolic blood pressure levels were decreased upon 16/24 weeks pioglitazone treatment. HDL cholesterol was increased after treatment with pioglitazone. The plasma creatinine level, insulin level, LDL cholesterol level, triglycerides level, systolic blood pressure and heart rate remained unaltered.
Patients with type 2 diabetes mellitus (T2DM) are known to have increased levels of HPSE activity, and it is well known that HPSE is essential for the development  of diabetic nephropathy. 18,37 Therefore, the plasma HPSE activity of T2DM patients was measured at baseline and compared with the HPSE activity level upon 16/24 weeks treatment with pioglitazone. Pioglitazone did not affect the plasma HPSE activity of the T2DM patients (P = 0.21) (Fig. 6a). However, a decrease in plasma HPSE activity can be observed in patients in the lowest quartile of the cohort, which can be appreciated when the lowest quartile of the baseline is compared with the lowest quartile after treatment with pioglitazone presented in the boxplot (P = 0.07) (Fig. 6a). It should be noted that the HPSE activity was measured in plasma, whereas a more pronounced effect of pioglitazone on HPSE activity might be observed in renal cortex. Similarly, a trend of decreased heparanase expression levels in plasma can be observed in the patient cohort after treatment with pioglitazone (P = 0.12) (Fig. 6b). One of the outcome parameters that was reported in the PIRAMID study to change significantly upon pioglitazone treatment is HbA 1C , 33 which describes the average blood glucose levels for the last two to three months. We observed higher HPSE activity levels in patients whose HbA 1C levels exceeded 6.5% (Fig. 6c), although no direct correlation could be observed between HPSE activity and HbA 1C (data not shown). HPSE activity did show a moderate, near significant correlation with plasma creatinine levels (Fig. 6d).

Discussion
In this study, we showed that PPARɣ regulates the expression of HPSE in cultured podocytes, cultured glomerular endothelial cells, healthy rats and in a rat model for human FSGS. In agreement with our previous studies, rats showed increased glomerular HPSE expression and reduced glomerular HS expression after induction of AN. 31,38 Treatment with the PPARɣ agonist pioglitazone normalized glomerular HPSE and HS expression, whereas PPARɣ antagonism induced glomerular HPSE expression and reduced glomerular HS expression. Importantly, in line with the induction of proteinuria in vivo, we showed that PPARɣ antagonism induced transendothelial albumin passage in vitro in a HPSE-dependent manner. In addition, we showed that the PPARɣ antagonist GW9662 induced HPSE promoter activity, suggesting that the transcription factor PPARɣ directly suppresses HPSE transcription.
The present study shows that PPARɣ is a negative regulator, i.e. repressor, of glomerular HPSE. A previous study revealed that PPARɣ inhibited hepatocellular carcinoma migration by downregulating pro-metastatic genes, including HPSE by direct binding to the HPSE promoter region. 14 We now show direct binding of PPARɣ to the HPSE promoter in mouse podocytes and glomerular endothelial cells. Adriamycin treatment significantly reduced the binding of PPARɣ to the HPSE Fig. 3: The PPARɣ ɣ antagonist GW9662 induced glomerular heparanase expression and reduced glomerular HS expression in mice. (a) Cortical heparanase mRNA expression, (b and e) glomerular heparanase protein expression, and (c) cortical heparanase activity were increased by treatment with 2.5 mg/kg body weight of the PPARɣ antagonist GW9662 for 3 weeks, whereas glomerular HS expression was reduced according to semi-quantitative analysis of immunofluorescence images (d and f). Representative pictures of (e) glomerular heparanase protein expression and (f) glomerular HS expression (magnification ×400). 4 rats per group were used for analysis. Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01. AU, arbitrary units. promoter in podocytes, and a similar trend was observed for glomerular endothelial cells. PPARɣ agonism inhibits the decline in PPARɣ binding to the HPSE promoter upon adriamycin treatment. In addition, treatment of podocytes and glomerular endothelial cells with the PPARɣ antagonist GW9662 tended to reduce PPARɣ binding to the HPSE promoter. These data suggest that in podocytes and most likely also in glomerular endothelial cells, PPARɣ represses gene expression potentially through co-repressors such as the nuclear receptor corepressor (NCoR) or the silencing mediator of retinoid and thyroid hormone receptors (SMRT) via histone deacetylases and subsequent transcriptional repression of HPSE. 39 Our in vitro results support the hypothesis that PPARɣ regulates HPSE gene expression. Although both mPC-5 and ciGEnC increased HPSE mRNA expression upon treatment with adriamycin, the HPSE mRNA expression from mGEnC-1 decreased upon adriamycin treatment after 16 h. Previously, we observed that the initial response of mGEnC-1 to adriamycin is an increased HPSE mRNA expression in the first hours after exposure, whereas HPSE mRNA expression is decreased after 16 h. However, the HS expression by mGEnC-1 is still decreased after 16 h of adriamycin treatment, corresponding to an increased albumin passage through mGEnC-1 monolayers.
PPARɣ agonists, like the TZDs pioglitazone and rosiglitazone, have already been clinically applied for a few decades. As they reduce insulin resistance, they are mainly used for the treatment of patients with type 2 diabetes. 4,5 However, a number of studies also suggest that PPARɣ agonists have direct renoprotective effects. 6,7,12,13 HPSE has been shown to be essential for the development of diabetic nephropathy, and type 2 diabetes patients show increased levels of HPSE activity. 18,37 We showed that plasma HPSE activity in these pioglitazone treated patients was related to their HbA1c level and correlating with serum creatinine levels. Even though no clear effect on plasma HPSE activity could be observed upon pioglitazone treatment, a decrease of HPSE activity in the renal cortex cannot be omitted. Since treatment with pioglitazone and rosiglitazone both reduced proteinuria in patients with non-diabetic renal diseases, 12,13,40 TZDs could be considered for treatment of proteinuria in nondiabetic renal diseases as well. A major drawback of TZDs is the development of severe side effects, such as fluid retention, edema formation, cardiac failure and an increased risk for bone fractures. 41 With this study, we provide further mechanistic in vitro and rat in vivo evidence that TZDs have direct renoprotective effects, by repressing glomerular HPSE expression and activity.
In addition to HPSE, several mechanisms have been described that may explain the renoprotective effects of PPARɣ agonists. A previous study showed that the PPARɣ agonist rosiglitazone partially reduced proteinuria by preserving the expression of the slit diaphragm proteins nephrin, podocin, and CD2AP in rats with AN. 9 More recently, we showed that PPARɣ agonists reduced the expression of the slit diaphragm protein transient receptor potential channel C6 (TRPC6) in podocytes. 11 Previous studies showed that glomerular TRPC6 expression is increased in several proteinuric diseases, suggesting that TRPC6 plays a role in the development of proteinuric diseases. 42 PPARɣ negatively regulated TRPC6 expression by direct binding to the TRPC6 promoter, and thereby reduced podocyte damage and the development of proteinuria in rats with AN. 11 In addition to the slit diaphragm proteins TRPC6, nephrin, podocin and CD2AP, PPARɣ also reduced the expression of transforming growth factor-β (TGF-β), endothelin-1 and the renin-angiotensin-aldosterone system (RAAS), and increased the bioavailability of nitric oxide. [43][44][45][46][47][48] Interestingly, all these aforementioned factors are involved in the regulation of HPSE as well. 38,[49][50][51] Together, there appear to be several mechanisms underlying the renoprotective effects of PPARɣ agonists.
There are also some limitations in our study. First, the patient samples are from T2DM patients, while pioglitazone is known to have a glucose-lowering effect and lower blood glucose level may affect HPSE expression and/or activity as well. 52,53 However, the animal model that was used in this study was not a diabetes model. Second, we only assessed HPSE expression levels but not the more relevant HPSE activity for the in vitro models because we were unable to measure this for the endothelial cells. Third, the route of administration of pioglitazone can be discussed; we have administered pioglitazone via i.p injection. Although pioglitazone is administered orally in patients, others have administered pioglitazone via i.p injection in animal studies as well. 54 Fourth, the use of cell lines in   mono-culture is not ideal, since it is well known that the cross-talk between podocytes and endothelial cells play a key role in disease manifestation. [55][56][57] Future experiments should thus focus on the development of cocultures or organ-on-a-chip technologies able to test the effects of e.g. PPARy agonism on such interactions. Finally, we only focused on the glomerular cells as possible source and target of HPSE, whereas it is well known that various cell-types, including non-glomerular endothelial cells and immune cells, can be a source of HPSE and various cell types and the extracellular matrix can be a target of HPSE. 53,58 Our current study provides an additional mechanism for the renoprotective effects of pioglitazone and other PPARɣ agonists. By reducing glomerular HPSE expression and activity, glomerular HS expression is preserved and the development of proteinuria is prevented. In the past, AN was mainly regarded as a podocyte damage model for human FSGS. However, more recent studies showed that glomerular endothelial cells play a crucial role in the development of AN, as the glomerular endothelial glycocalyx thickness is reduced by 80% in mice with AN, and HS expression on cultured mouse glomerular endothelial cells is reduced by Adriamycin. 31,59 Additionally, a recent study showed that endothelial cell damage precedes podocyte damage in AN, 60 further highlighting the importance of the glomerular endothelium in the development of AN. Our current data suggest that PPARɣ agonists have direct protective effects on both glomerular endothelial cells and podocytes, by reducing HPSE expression and transendothelial albumin passage.
In conclusion, our study suggests that PPARɣ agonists like pioglitazone reduce proteinuria by inhibiting glomerular HPSE expression, thereby providing an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice.  DHvR, RWvdM and HJL provided the patient materials. All authors revised and approved the final version of the manuscript.

Data sharing statement
This study did not generate any new unique reagents, datasets or code. The main data supporting the findings of this study are available within the paper. Further information and requests for resources and reagents should be directed to the corresponding author.

Declaration of interests
The authors declare that there are no conflicts of interest.